JP2007322378A - Blood type determination method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a blood type determination method capable of determining easily and accurately a blood type. <P>SOLUTION: This blood type determination method for inspecting a hemocyte includes (i) a process for solidifying the hemocyte on a substrate, (ii) a process for making a labeled antibody coupled specifically with an antigenic portion expressing the blood type of the hemocyte react with the hemocyte, before or after the process (i), (iii) a process for photographing microscopically the solidified hemocyte and for recognizing the individual cells to prepare a cell distribution pattern, (iv) a process for photographing microscopically the labeled antibody coupled to the solidified hemocyte and for preparing a distribution pattern of the labeled antibodies, and (v) a process for determining reactivity of the hemocyte with the labeled antibody, based on the cell distribution pattern prepared in the process (iii), and based on the labeled antibody distribution pattern prepared in the process (iv). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a blood type determination method, and more particularly, to a method for determining a blood type by detecting an antigen-antibody reaction in individual cells using a microscope.

  Conventionally, an agglutination method has been used for determining a blood type by observing the aggregation of blood cells (for example, Patent Document 1). However, the measurement by the agglutination method has a problem that it is difficult to quantify the test results and lacks objectivity.

  As another method of blood type determination, there are solid phase methods such as EIA method and MPHA method. In the solid phase method, cells are solid-phased on a substrate or well, a fluorescently labeled antibody or an antibody with particles attached thereto is reacted, fluorescence or light transmittance is measured, and determination is made based on the distribution pattern of fluorescence or particles. However, the number of cells immobilized on a substrate or a well is not constant, and variation occurs from sample to sample, resulting in a problem that measurement error increases.

Another method for determining the blood type is a method using a flow cytometer (FCM). The method using FCM has the advantage that the antigen-antibody reaction of individual cells can be measured. However, since it is necessary to perform B / F separation in order to use the specimen for FCM, there is a problem that the operation is complicated and is not suitable for a case where it is necessary to process many specimens.
JP 2001-4625 A

  In view of the above problems, an object of the present invention is to provide a blood type determination method capable of easily performing accurate blood type determination.

  To achieve the above object, the present invention provides a blood group determination method characterized by examining blood cells by the following steps: (i) immobilizing blood cells on a substrate; ii) before or after the step (i), reacting a labeled antibody capable of specifically binding to the antigenic site representing the blood type of the blood cell with the blood cell, and (iii) immobilizing the solid phase. A step of microscopically photographing the formed blood cells, recognizing individual cells to create a cell distribution pattern, and (iv) photographing the labeled antibody bound to the solid-phased blood cells with a microscope. A step of creating a distribution pattern; (v) a reaction of the blood cell to the labeled antibody based on the cell distribution pattern created in step (iii) and the labeled antibody distribution pattern created in step (iv) The process of determining sex.

  In another embodiment of the present invention, there is provided a blood type determination method characterized by examining an antibody by the following steps: (a) a substrate containing antigen cells that can specifically bind to an antibody representing a blood group (B) a step of reacting a sample solution containing the antibody to be examined with the immobilized antigen cells, and (c) an antibody bound to the immobilized antigen cells. A step of further binding a labeled antibody that specifically binds to the antibody, and (d) taking a micrograph of the immobilized antigen cell, recognizing each cell, and creating a cell distribution pattern And (e) microscopically photographing the labeled antibody further bound to the antibody bound to the antigen cell to create a distribution pattern of the labeled antibody, and (f) the cell distribution created in the step (d). Pattern and labeled antibody prepared in step (e) Based on the fabric pattern, step of determining reactivity to the antigenic cells of the test subject antibody.

  In the above method, the step of microscopically imaging the solid-phased cells and recognizing individual cells to create a cell distribution pattern identifies the cells by size and specifies the position of each cell. It is preferable that the process is performed, and it is particularly preferable to use a microscopic image in a bright field.

  According to the blood type determination method of the present invention, the antigen-antibody reaction of individual cells can be detected very simply by using a microscope, and therefore accurate blood type determination can be easily performed.

  According to a first aspect of the present invention, a method for frontal testing of examining blood cells is provided.

  Here, the blood cell refers to a cell component in blood, and includes red blood cells, white blood cells, and platelets. There are various blood types such as ABO blood type, S blood type, Rh blood type, MN blood type, blood type related to HLA antigen (human leukocyte antigen), blood type related to HPA antigen (human platelet antigen), etc. However, the blood type determination method of the present invention can be applied to any blood type determination.

  In the blood type determination method in the first embodiment, first, blood cells are immobilized on a substrate, and then a labeled antibody that can specifically bind to an antigenic site representing the blood type of the blood cells is reacted.

  The substrate used here is made of a material suitable for solidifying cells, and any substrate can be used as long as it can be used for microscopic photography, and a glass substrate is preferably used. Cell immobilization can be performed by methods well known in the art.

  As the labeled antibody, an antibody corresponding to the antigen in the blood group to be measured is used. For example, when testing the ABO blood group, the antigens are erythrocyte type A and type B antigens, and an A antigen-specific antibody and a B antigen-specific antibody are used.

  As the labeled antibody, one labeled in advance with a labeling substance such as a fluorescent labeling substance, a radioisotope, or a dye is used, but it is preferable to label with a fluorescent labeling substance. The fluorescent labeling substance may be, but is not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) and the like that are usually used in the art.

  If there are multiple antibodies, they can be tested simultaneously by labeling each antibody with a different labeling substance. For example, in the case of an ABO blood group test, an A antigen-specific antibody is labeled with FITC, and a B antigen-specific antibody is labeled with TRITC. Thus, as shown in FIG. 1, erythrocytes to which neither label binds are of type O, only FITC-labeled antibodies bind to type A, and only TRITC-labeled antibodies bind to type B. It can be seen that both antibodies bind to the AB type. Thus, by labeling each antibody differently, it is not necessary to test each antibody, and a plurality of antibodies can be used simultaneously.

  The reaction between the labeled antibody and the blood cell may be performed under a condition that causes an antigen-antibody reaction, and may be performed by dropping a sample solution containing the labeled antibody onto the blood cell immobilized on the substrate.

  The reaction between the labeled antibody and the blood cell may be performed before immobilizing the blood cell on the substrate, and the blood cell may be immobilized after the reaction.

  After the reaction between blood cells and labeled antibody, the substrate is washed to remove unreacted labeled antibody. In the method of the present invention, since the cells are immobilized, the unreacted labeled antibody can be easily removed simply by washing.

  Next, the solidified blood cells are photographed under a microscope to recognize individual cells and create a cell distribution pattern. That is, the cells existing on the substrate and the contaminants other than the cells are identified, and the cells are confirmed and their positions are recorded. The cell distribution pattern is for recording at which position on the substrate the cells are solid-phased, and may be created by analyzing an image taken with a microscope. For example, the cell may be identified according to its size, and the cell distribution pattern may be determined by specifying the position of each cell using the coordinate axis on the substrate.

  At the time of microscopic photography, the cells may be stained with a conventionally known dye, or may be detected by binding a label different from the antibody. However, since these operations are complicated, it is preferable to perform microscopic photography of cells in a bright field, and to identify cells and create distribution patterns from the images.

  Separately from the creation of the cell distribution pattern, the labeled antibody bound to the blood cells immobilized on the substrate is photographed under a microscope, and the distribution pattern of the labeled antibody is created. In this method, the labeling substance bound to the antibody is analyzed by microscopic imaging using a method suitable for the labeling substance, thereby creating the position of the labeling antibody present on the substrate as a distribution pattern. For example, when an antibody is labeled with a fluorescent labeling substance, the image may be taken using appropriate excitation light and a filter.

  FIG. 2 shows a conceptual diagram of each pattern. FIG. 2A is a schematic diagram when a cell is photographed in a bright field. The cell is recognized and its position is confirmed. FIG. 2 (b) is a diagram of fluorescence imaging in a dark field under the detection condition of FITC label, and shows that the cells emit fluorescence. On the other hand, FIG. 2 (c) is a schematic diagram when the image is taken under the detection conditions of TRITC label, and shows that the cell does not have fluorescence and the TRITC-labeled antibody is not bound. Accordingly, the cells in FIG. 2 are found to be cells to which only the FITC-labeled antibody binds.

  The preparation of the labeled antibody distribution pattern may be performed before or after the preparation of the cell distribution pattern, but may be performed in parallel. For example, in a certain visual field, a cell distribution pattern is created, and then the labeled antibody distribution pattern is imaged by photographing under conditions suitable for each labeled antibody. Then, the visual field may be moved and the same process may be repeated again to create a pattern for the entire substrate.

  Finally, by comparing the created cell distribution pattern with the labeled antibody distribution pattern, the presence / absence of binding between the cells and the labeled antibody, the labeled signal intensity of one cell, and the like are analyzed. Among cells solid-phased on a substrate, cells having a label having a predetermined intensity or more are present in a predetermined ratio or more, so that the cells can be regarded as cells that bind to the labeled antibody. That is, it can be determined that the antibody has antigenicity against the labeled antibody, and the blood type is determined from the result.

  Next, a blood type determination method for a back test for examining antibodies according to the second aspect of the present invention will be described.

  Antibodies that represent blood groups are present in serum, blood, and other body fluids, and include, for example, antigens representing various blood groups such as ABO blood group, S blood group, Rh blood group, and MN blood group. Includes antibodies that specifically bind.

  In the method of this embodiment, first, antigen cells that may specifically bind to the antibody to be examined representing the blood group are immobilized on a substrate. For example, when testing an ABO blood group, the antibodies to be tested are an A antigen-specific antibody and a B antigen-specific antibody, and erythrocytes having an A antigen and a B antigen that can specifically bind to these antibodies are used as antigens. Used as a cell. If there are multiple antigen cells, they are immobilized separately.

  Next, the sample solution containing the antibody to be detected is applied to the immobilized antigen cells and allowed to undergo an antigen-antibody reaction under appropriate conditions. After the reaction is completed, unreacted antibody is washed away.

  Next, the sample solution containing the labeled antibody is applied to the substrate, and the labeled antibody is further bound to the antibody bound to the immobilized antigen cell under appropriate conditions. After the reaction between the antibody and the labeled antibody is completed, the unreacted labeled antibody is removed by washing.

  The labeled antibody used here is a so-called secondary antibody, and an antibody that specifically binds to the antibody to be examined is labeled with a labeling substance and used. The labeled antibody may be, for example, immunoglobulin (Ig), and the labeling substance may be the same as that described in the first embodiment, but it is particularly preferable to use a fluorescent labeling substance.

  FIG. 3 is a conceptual diagram of this aspect. The antibody to be detected binds to the antigenic site of the antigen cell immobilized on the substrate, and the labeled Ig antibody binds to this antibody.

  In FIG. 3, anti-human IgG antibody and anti-human IgM antibody were used as labeled antibodies. For example, when testing an ABO blood group, there are two types of human Ig antibodies that react with erythrocytes, IgG antibodies and IgM antibodies, and the ratios thereof vary from individual to individual. Therefore, by using an anti-human IgG antibody and an anti-human IgM antibody as labeled antibodies and labeling them with the same labeling substance, accurate measurement can be performed regardless of which human Ig antibody the antibody contained in the sample to be examined is. Is possible.

  Next, the immobilized antigen cells are photographed under a microscope, and a distribution pattern of individual cells is created as described in the first embodiment.

  In addition to the preparation of the cell distribution pattern, the labeled antibody further bound to the antibody bound to the antigen cell is photographed under a microscope, and the distribution pattern of the labeled antibody is created as described in the first embodiment.

  Finally, by comparing the created cell distribution pattern with the labeled antibody distribution pattern, the presence / absence of binding between the antibody to be detected and the antigen cell, the labeled signal intensity of one cell, and the like are analyzed. It is possible that the antibody to be tested has reactivity with the antigen cells used because of the presence of a predetermined percentage or more of cells having a label having a predetermined intensity or more among the cells immobilized on the substrate. From this result, the blood type is determined.

  As described above, according to the blood type determination method of the present invention, since the antigen-antibody reaction of individual cells can be detected, accurate determination is performed without being affected by the number of immobilized cells. It is possible. In addition, since the cells immobilized on the substrate are photographed using a microscope, the unreacted labeled antibody can be removed simply by washing, and it is not necessary to perform B / F separation. Can be implemented.

The conceptual diagram of the blood type determination by a labeling substance. The conceptual diagram which shows the distribution pattern of a cell and a labeled antibody. The conceptual diagram of the antigen antibody reaction in a 2nd aspect.

Claims (4)

A blood type determination method characterized by examining blood cells by the following steps:
(I) immobilizing blood cells on a substrate;
(Ii) before or after the step (i), reacting a labeled antibody capable of specifically binding to an antigenic site representing the blood type of the blood cell with the blood cell,
(Iii) microscopically photographing the solid-phased blood cells, recognizing individual cells and creating a cell distribution pattern;
(Iv) microscopically imaging the labeled antibody bound to the solid-phased blood cell, and creating a distribution pattern of the labeled antibody;
(V) A step of determining the reactivity of the blood cells to the labeled antibody based on the cell distribution pattern created in the step (iii) and the labeled antibody distribution pattern created in the step (iv). A blood type determination method characterized by examining antibodies by the following steps:
(A) immobilizing an antigen cell capable of specifically binding to an antibody representing a blood group on a substrate;
(B) reacting a sample solution containing the antibody to be examined with the immobilized antigen cells;
(C) further binding a labeled antibody that specifically binds to the antibody bound to the immobilized antigen cell;
(D) microscopically imaging the immobilized antigen cells, recognizing individual cells and creating a cell distribution pattern;
(E) microscopically imaging the labeled antibody further bound to the antibody bound to the antigen cell, and creating a distribution pattern of the labeled antibody;
(F) A step of determining the reactivity of the antibody to be tested to the antigen cells based on the cell distribution pattern created in the step (d) and the labeled antibody distribution pattern created in the step (e).   The step of microscopically photographing the solid-phased cells and recognizing individual cells to create a cell distribution pattern is a step of identifying cells by size and specifying the position of each cell. The method according to claim 1 or 2.   The method according to claim 3, wherein the step of microscopically imaging the solid-phased cells and recognizing individual cells to create a cell distribution pattern is performed using a microscopic image in a bright field.

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